Botulinum toxin is the microbial agent responsible for the disease botulism. This toxin produces paralysis by inhibiting the release of acetylcholine from nerve endings. Botulinum toxin is also used clinically to treat neuromuscular diseases characterized by hyperactivity. The long-range goal of the proposed research is to define the molecular targets that mediate the mechanism of action of botulinum toxin at the clinically important toxin target tissue, the neuromuscular junction. To produce its toxic effect, botulinum toxin must first bind to specific receptors on the plasma membrane of cholinergic nerve terminals to gain entry to the intracellular space. Once inside, the toxin must find its intracellular target and inactivate it proteolytically. Although several membrane and intracellular synaptic proteins have been implicated as putative toxin receptors and intracellular targets in studies of non-target tissues, their identities at the neuromuscular junction are unknown. To begin to address these unknowns definitively, functional studies of toxin action must be combined with biochemical and molecular studies in the clinically relevant target tissue, the neuromuscular junction. To begin to resolve more fully the cellular and biochemical interactions of botulinum toxin with its target tissue, the proposed research will consist of two major elements. First, immunological detection techniques will be combined with electrophysiological recordings of endplate activity in mouse neuromuscular tissue to determine biochemically and functionally the interactions are botulinum toxin with putative intracellular target proteins. Second, the technique of molecular biology will be combined with electrophysiology to define at the molecular and functional level the specific target receptor responsible for the selective interaction of botulinum toxin with nerve terminals of the mouse neuromuscular junction. The results of the proposed studies will hopefully impact on clinical medicine by defining specific cellular targets of the neuromuscular junction that may serve as molecular templates for the development of more effective toxin antagonists and safer toxin-like therapeutic agents.